Cell-bound cations of the moderately halophilic bacterium Vibrio costicola.

Over most of the range of salt concentrations in which the moderately halophilic bacterium Vibrio costicola could grow, the sum of the cell-associated Na+ + K+ ions was at least as high as in the external medium. This is in contrast to other moderate halophiles, which have substantially lower internal than external salt concentrations for most of their growth range. The relative amounts of Na+ and K+ in V. costicola varied with environmental conditions. The K+/Na+ ratio fell during anaerobic incubation or when cells were poisoned. As Na+ ions left the cells, K+ ions entered. However, movement of these ions was not tightly coupled, since K+ content of cells could increase without a corresponding decrease in Na+ content. The Mg2+ contents of cells varied little with environmental conditions.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).     

Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola.

The interactive effects of NaCl concentration and growth temperature on the growth and lipid composition of the moderately halophilic eubacterium Vibrio costicola have been investigated. Vibrio costicola was shown to be capable of growth over the temperature range 4-37 degrees C. Maximum growth yields were obtained at 30 degrees C when the optimum NaCl concentration was 1.0 M NaCl. In contrast with some previous studies, at higher or lower growth temperatures both the optimum and lower limit of NaCl concentration were higher, but there was no change in the upper limit of NaCl concentration for growth. There were no differences between the lipid compositions of cultures grown in 1 M NaCl at 30 or 37 degrees C, but as the growth temperature was lowered from 30 to 10 or 4 degrees C, the ratio of phosphatidylethanolamine to phosphatidylglycerol increased significantly as a result of the conversion of phosphatidylglycerol to diphosphatidylglycerol; in addition, at the lower growth temperatures the phospholipid fatty acyl composition became more unsaturated and the mean acyl chain length was shorter. It is suggested that the altered salt dependence of V. costicola at temperatures below the optimum for growth is due to a modification in membrane lipid phase behavior and stability brought about by changes in lipid composition, whereas a different mechanism operates above the growth temperature optimum.     

Isolation and characterization of salt-sensitive mutants of the moderately halophilic bacterium Salinivibrio costicola subsp. yaniae

Salinivibrio costicola subsp. yaniae is a moderately halophilic bacterium which can grow over a wide range of salinity. In response to external osmotic stress (1-3 M NaCl), S. costicola subsp. yaniae can accumulate ectoine, glycine betaine, and glutamate as compatible solutes. We used suicide plasmids pSUP101 to introduce transposon Tn1732 into S. costicola subsp. yaniae via Escherichia coli SM10 mediated by conjugation. One Tn1732-induced mutant, MU1, which was very sensitive to the external salt concentration, was isolated. Mutant MU1 did not grow above 1.5 M NaCl and did not synthesize ectoine, but accumulated Ngamma-acetyldiaminobutyrate, an ectoine precursor, as confirmed by (1)H-NMR analysis. From these data, we concluded that ectoine performs a key role in osmotic adaptation towards high salinity environments in strain S. costicola subsp. yaniae.     

A bacteriophage of a moderately halophilic bacterium

A bacteriophage of an aerobic, gram-negative, rod-shaped halophilic bacterium, provisionally named Pseudomonas sp. G3, is described. The phage has a head and a tail and is similar in appearance to Salmonella phage Beccles. It infects its bacterial host at all salt concentrations in which the bacteirum is able to grow. In contrast to phages of halophilic archaebacteria, the newly-described phage is relatively stable in the absence of salt. It also infects Vibrio costicola and two unidentified halophilic eubacteria.Abbreviations PPT proteose peptone-tryptone medium - pfu plaque-forming unit - G+C guanine + cytidine content, mol %     

Isolation of salt-sensitive mutants of the moderately halophilic eubacteriumVibrio costicola

A protocol is described for the first isolation of salt-sensitive mutants from a moderately halophilic eubacterium, namelyVibrio costicola, which normally can grow in up to 3.4M NaCl. Four groups of mutants, which are all unable to grow at high (i.e., 2.5–3.0M) concentrations of NaCl, have been characterized on the basis of their salt and temperature sensitivities. The acquisition of these mutants will be helpful in clarifying the role of membrane lipid changes in haloadaptation.     

Characterization of halophilic alkaline phosphatase from Halomonas sp. 593, a moderately halophilic bacterium

A halophilic alkaline phosphatase was highly purified (about 510-fold with about 21% yield) from a moderate halophile, Halomonas sp. 593. The N-terminal 35 amino acid sequence of this enzyme was found to be more acidic than those previously isolated from Vibrio spp., and this enzyme was partially resistant to SDS. Several enzymatic properties demonstrated that it showed higher halophilicity than those enzymes from Vibrio spp.     

Growth of a marine Vibrio alginolyticus and moderately halophilic V. costicola becomes uncoupler resistant when the respiration-dependent Na+ pump functions

The growth of Vibrio alginolyticus and V. costicola, which possess respiration-dependent Na+ pumps, was highly resistant to the proton conductor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), in alkaline growth media, even though the membrane was rendered permeable to H+. The pH dependence of CCCP-resistant growth was similar to that of the Na+ pump. In contrast, Escherichia coli ML308-225 showed neither Na+ pump activity nor CCCP-resistant growth, even when grown in alkaline, Na+-rich media. These results suggest that certain bacteria possess the Na+ pump and are thus able to grow under the conditions where H+ circulation across the membrane does not take place. Moreover, V. alginolyticus growing in the presence of CCCP maintains normal levels of internal K+, Na+, and H+. The Na+ pump, therefore, makes the growth of these organisms resistant to CCCP by maintaining the intracellular cation environments.     

Oceanobacillus aidingensis sp. nov., a moderately halophilic bacterium

Two Gram-positive, rod-shaped moderately halophilic bacterial strains, designated AD7-25^T and AB-11, were isolated from Aiding and Manasi salt lakes in Xinjiang of China, respectively. The strains were found to be able to grow at NaCl concentrations of 0–21 % (w/v), with optimum growth occurring at 6–8 % (w/v) NaCl. The optimal temperature and pH for growth were determined to be 33–37 °C and pH 7.0–7.5. Cells of the strains are motile by means of polar flagella. Both strains can produce ellipsoidal spores. The major cellular fatty acids were identified as anteiso-C_15:0, iso-C_15:0, iso-C_14:0, anteiso-C_17:0 and iso-C_16:0. The diamino acid in the peptidoglycan and the major quinone system were determined to be meso-diaminopimelic acid (meso-DAP) and MK-7, respectively. The DNA G+C contents of stains AD7-25^T and AB-11 were 39.8 and 40.0 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that these two novel strains are closely related to the genus Oceanobacillus showing 90–99.5 % similarity with respect to type strains. These two novel strains were most closely related to Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557^T (99.1 and 99.5 %), followed by O. oncorhynchi subsp. oncorhynchi JCM 12661^T (99.1 and 99.4 %), Oceanobacillus neutriphilus CGMCC 1.7693^T (97.0 and 97.5 %), Oceanobacillus sojae JCM 15792^T (97.6 and 98.0 %) and Oceanobacillus locisalsi KCTC 13253^T (96.5 and 96.9 %). The DNA–DNA hybridization data indicated that DNA relatedness between strains AD7-25^T and AB-11 was 91.0 %, and the genomic homology of representative strain AD7-25^T with O. oncorhynchi subsp. incaldanensis DSM 16557^T, O. oncorhynchi subsp. oncorhynchi JCM 12661^T, O. neutriphilus CGMCC 1.7693^T, O. sojae JCM 15792^T and O. locisalsi KCTC 13253^T were 41, 39, 20, 23 and 17 %, respectively. On the basis of phenotypic and phylogenetic distinctiveness, strains AD7-25^T and AB-11 should be assigned to the genus Oceanobacillus as a new species, for which the name Oceanobacillus aidingensis sp. nov. was proposed. The type strain is AD7-25^T (=CGMCC 1.9106 ^T = NBRC 105904^T).     

The moderately halophilic bacterium Halomonas maura is a free-living diazotroph

Halomonas maura is a moderately halophilic bacterium which lives in saline soils and synthesises an exopolysaccharide known as mauran. Strain S-31T grew in a nitrogen-free medium under an N2 atmosphere; the acetylene reduction assay proved positive under specific conditions. We identified the nifH gene in this strain by using degenerate oligonucleotides designed from highly preserved gene sequences obtained from the alignment of a large number of nifH sequences from different microorganisms. Our results lead us to conclude that H. maura is capable of fixing nitrogen under microaerobic conditions.     

Lipids and fatty acids of a moderately halophilic bacterium, No. 101.

The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaC1, were examined. The extractable lipids were separated into at least 8 components by using thin-layer chromatography. The major phospholipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phosphoglycolipid in the whole cells, cell envelopes and outer membrane preparations, commonly. Judging from mild alkaline hydrolysis and exzymatic treatment with phospholipase A2, C and D, the unidentified phosphoglycolipid possessing Pi, glycerol, fatty acids and glucose in a molar ratio of 1 : 2 : 2 : 1, appeared likely to be a glucosyl derivative of phosphatidylglycerol. No glucuronic acid containing lipid was detected. The exractable lipid composition varied greatly with the concentrations of NaC1 in the medium and the stages of bacterial growth. The most characteristic phosphoglycolipid in this organism increased up to 25% of the total phospholipids with the addition of 1% glucose in the medium. The major fatty acids of the extractable lipids were C16:0, C16:1, C18:0, C18:1 and cyclopropanoic C17 and C19 acids and these compositions were very similar for each phospholipid. The cyclopropanoic fatty acids predominated as growth proceeded. The fatty acids of the bound lipids comprised a high concentration of 3-hydroxydodecanoic acid. The esterified fatty acids of the lipopolysaccharide molecule seemed to contain a wide variety of hydroxy and non-hydroxy shorter chain fatty acids, while the amide-linked fatty acids consisted almost entirely of 3-hydroxydodecanoic acid.     

Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola

Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.     

一株中度嗜盐芽孢杆菌(Bacillus sp. BZ-SZ-XJ39)的微生物学特性

【目的】了解一株来自新疆盐碱湖的中度嗜盐菌(Bacillus sp.BZ-SZ-XJ39)的微生物学特性,为进一步阐明该菌独特的盐适应机制奠定基础。【方法】通过16S rRNA基因序列分析、G+C mol%含量测定、细胞形态和菌落观察、培养条件确定、营养与生理生化指标测定以及细胞化学组分分析等描述该菌的生物特性。【结果】基于16S rRNA基因序列的同源性和系统发育树分析,菌株BZ-SZ-XJ39与Bacillus saliphilus 6AGT(序列相似性为97.5%),Bacillus daliensis DLS13T(96.5%),Bacillus luteus JC167T(96.2%),Bacillus chagannorensis CG-15T(95.5%)和Bacillus agaradhaerens DSM8721T(95.3%)有密切的亲缘关系。基因组DNA G+C含量为44.4 mol%±1.2 mol%(Tm)。菌株BZ-SZ-XJ39为好氧、短杆状、革兰氏阳性菌。菌落呈黄色、圆形凸起、表面光滑且边缘整齐。菌株生长盐度范围为0.5%–28%(最适8%),pH范围为5.5–9.5(最适pH 8.0),温度范围为4–41oC(最适33oC)。菌株BZ-SZ-XJ39合成的主要脂肪酸包括anteiso-C15:0(50.2%)和anteiso-C17:0(16.3%)。合成极性脂为磷脂(PL)、磷脂酰乙醇胺(PE)、磷脂酰甘油(PG)、二磷脂酰甘油(DPG)和磺酸基异鼠李糖基二脂酰基甘油(SQDG)。呼吸醌类型为甲基萘醌(MK-7)。菌株BZ-SZ-XJ39已经在中国普通微生物菌种保藏管理中心(CGMCC1.12936)和日本微生物菌种保藏中心(JCM30194)冻干保藏,在Gen Bank中的序列注册号为KP456019。【结论】基于BZ-SZ-XJ39菌株的遗传型、表型、生理生化以及化学组成特征,初步确定该菌为Bacillus属中的一个新成员。研究将为探索生命在高盐环境中存在的本质提供新素材,也可为生物技术的潜在应用提供新视角。     

Salimicrobium salexigens sp. nov., a moderately halophilic bacterium from salted hides

Two Gram-positive, moderately halophilic bacteria, designated strains 29CMI^T and 53CMI, were isolated from salted hides. Both strains were non-motile, strictly aerobic cocci, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–12.5% [w/v] NaCl), between pH 5.0 and 10.0 (optimal growth at pH 7.5) and at temperatures between 15 and 40 °C (optimal growth at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed a similarity of 98.7% and were closely related to species of the genus Salimicrobium, within the phylum Firmicutes. Strains 29CMI^T and 53CMI exhibited 16S rRNA gene sequence similarity values of 97.9–97.6% with Salimicrobium album DSM 20748^T, Salimicrobium halophilum DSM 4771^T, Salimicrobium flavidum ISL-25^T and Salimicrobium luteum BY-5^T. The DNA G+C content was 50.7 mol% and 51.5 mol% for strains 29CMI^T and 53CMI, respectively. The DNA–DNA hybridization between both strains was 98%, whereas the values between strain 29CMI^T and the species S. album CCM 3517^T, S. luteum BY-5^T, S. flavidum ISL-25^T and S. halophilum CCM 4074^T were 45%, 28%, 15% and 10%, respectively, showing unequivocally that strains 29CMI^T and 53CMI constitute a new genospecies. The major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0, iso-C_15:0 and iso-C_14:0. The main respiratory isoprenoid quinone was MK-7, although small amounts of MK-6 were also found. The polar lipids of the type strain consist of diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid and one glycolipid. The peptidoglycan type is A1γ, with meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of the phylogenetic analysis, and phenotypic, genotypic and chemotaxonomic characteristics, we propose strains 29CMI^T and 53CMI as a novel species of the genus Salimicrobium, with the name Salimicrobium salexigens sp. nov. The type strain is 29CMI^T (=CECT 7568^T = JCM 16414^T = LMG 25386^T).